Regulation of pH is essential to the life of all living cells. For instance, the pH of the cytosol must be maintained near neutrality by extrusion of acid generated as a result of metabolism. The pH of lysosomes must be acidic to increase the activity of hydrolytic enzymes. The pH of mitochondria is alkaline. Movement of protons in these organelles are required for production of ATP. Recent evidence indicates that changes in the pH in the cytosol may regulate numerous cellular activities. Changes in pH may be required for initiation of growth of quiescent, non-dividing cells. Alterations in intracellular pH may also dictate the pathway of differentiation selected by developing cells. Local changes in pH may contribute to movements of cells, such as locomotion and division. Techniques for measurement of pH have been developed that employ the pH dependence of fluorescence emitted by fluorescent probes that have been incorporated into living cells. A microspectrofluorometer can be used to continuously record the pH and its changes in local regions of single living cells. In the present proposal, the microspectrofluorometer will first be used to determine the accuracy and precision of fluorometric measurements of intracellular pH in the egg of the Medaka fish and in amoeba in the cellular slime mold, Dictyostelium discoideum. The changes in pH in the Medaka egg will be determined, localized and correlated with ongoing developmental processes. Changes in intracellular pH during development in D. discoideum will be analyzed in detail to test the hypothesis that changes in pH contribute to cell differentiation. Mechanisms or regulation of intracellular pH will be assessed by use of specific pharmacologic agents. In addition, changes in intracellular pH during cell movement in D. discoideum will be examined in order to test the hypotesis that pH is a regulator of the structure of cytoplasm, and the shape and movements of cells. Cell movements and differentiation are fundamental processes in the life of all vertebrate organisms. Elucidation of the role of pH in regulating these processes will advance our knowledge of the fundamental mechanisms that regulate cell activities, direct future studies of these phenomena in normal and pathologic states, and may eventually contribute to development of diagnostic procedures.